Vitamin B12 (cobalamin) is among the largest known non-polymeric natural products and is a cofactor that is synthesized only by certain prokaryotes. Among the bacteria that produce and utilize B12 is the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Recently, a novel enzyme, BluB, was discovered in S, meliloti and shown to catalyze the biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of B12 whose biosynthesis was previously unknown. BluB catalyzes the fragmentation of flavin mononucleotide (FMN) to form DMB in an oxygen-dependent reaction. This proposal aims to dissect the mechanism of this highly unusual enzyme by pre-steady state kinetics combined with genetic analyses. Additionally, this proposal seeks to understand the parallel but unrelated DMB biosynthetic pathway utilized by anaerobic bacteria. The anaerobic pathway is hypothesized to branch from the purine biosynthetic pathway rather than using FMN as a precursor. Candidate genes involved in this pathway will be identified by bioinfonnatics and tested by genetic and biochemical methods. The proposal also aims to understand the regulation and physiological significance of a novel DNA damage response in S. meliloti that occurs when B12 is limiting. This pathway controls the production of an altered exopolysaccharide. The genes that mediate this response will be identified in a screen for altered expression of genes involved in exopolysaccharide production. Subsequently, other targets of this regulatory pathway will be identified. Together these experiments will contribute to the understanding of the biosynthesis and function of B12 in bacteria and may lead to advances in human nutrition and disease treatment.